This application addresses broad Challenge Area (14) Stem Cells and specific Challenge Topic, 14-AG-105 Exploratory studies of induced pluripotent stem (iPS) cells from healthy individuals and patients with mental/nervous system disorders. Chromatin insulators are large DNA protein complexes that play a role in regulating gene expression. The structure, composition and function of these complexes is well understood in Drosophila but much less information is available in mammals. Work in our laboratory has contributed in large part to the current view of the role of insulators in nuclear function i.e. the three-dimensional organization of the chromatin fiber within the nucleus. A critical issue now is to determine whether this organization is important for the establishment and/or maintenance of patterns of gene expression that drive cell differentiation. We propose experimental strategies to understand the molecular mechanisms controlling nuclear organization during the process of stem cell differentiation. Given the possible importance of iPS cells to human health, it is critical also to understand all epigenetic changes that take place when differentiated cells are reprogrammed into iPS cells, including changes in nuclear organization. Preliminary results in our lab suggest that the organization of CTCF insulators in mouse stem cells changes as these cells differentiate. In addition, a wealth of information has become recently available describing the genomic localization of the CTCF protein in a variety of cell types. Interactions between these genomic CTCF sites determine the pattern of organization of the DNA in the nucleus. Here we propose to determine the three-dimensional structure of a region of the genome in cells at different states of differentiation. This will be accomplished using 5C to determine all possible interactions between a subset of CTCF sites and analyzing the data using computational methods derived from those used to analyze NMR protein data. The 5C experiments will be carried out with mouse stem cells, neural progenitor cells, neural-derived iPS cells and dopaminergic neurons. Results will shed light on the epigenetic differences and similarities between stem, differentiated and iPS cells. PUBLIC HEALTH RELEVANCE: The arrangement of the DNA of in the nucleus of eukaryotic cells is important in the regulation of gene transcription. DNA sequences called insulators and associated proteins are important in establishing the pattern of DNA organization in the nucleus. We propose to study how insulators contribute to changes in DNA organization taking place when stem cells become differentiated cells and when these cells are re-programmed to become iPS cells.